The objective of this project is to study the mechanism of action of the enzyme aspartate aminotransferase by site-directed mutagenesis. About 6 different amino acids at the active site will be replaced in separate experiments. The mutated enzymes will be examined by an appropriate combination of spectroscopic and kinetic methods. The results will provide information on the relative barrier heights facing the intermediates along the reaction pathway, will help to assign the ionizable groups which are responsible for the inflections in the kinetic pH profiles, and will provide information about the roles of several amino acids in stabilizing intermediates or transition states. A significant aspect of this choice of enzyme is the variety of probes that can be used in combination to examine each mutant. The ubiquity of PLP-dependent coenzymes in amino acid metabolism makes it likely that many of the results of this study will have bearing on the broader problem of the mechanism of action of the entire class. An additional goal of this research that is unrelated to the above, is to design a so-called suicide inhibitor for Beta-lactamase, based on the interaction of a postulated active site enzyme carboxylate moiety with the side-chain of Alpha-cyano benzylpenicillin. The ketenimine formed by general base catalyzed abstraction of the Alpha-proton is a very active electrophilic trapping agent. Success with this inhibitor might lead to a new class of pharmacologically useful inhibitors of Beta-lactamases that could be used in conjunction with penicillins or cephalosporins to increase the efficacy of penicillin-based chemotherapy.